This invention relates to human cytomegalovirus (HCMV), and is concerned with the production of glycoproteins of the virus, their vaccine potential, and the production of HCMV specific antibodies.
HCMV is a human pathogen of considerable importance and there is a demand for an effective vaccine against it. Hitherto experimental vaccines have been based on attenuated, non-pathogenic forms of the virus, but these can have undesirable side effects. The invention provides an alternative approach to the production of a vaccine against HCMV, using recombinant DNA techniques.
Like other herpes viruses HCMV specifies multiple glycoproteins (1,2). Characterisation of these have involved studies of CMV-infected cells and purified virions using polyclonal sera and monoclonal antibodies (2-10). One glycoprotein has been partially purified and shown to elicit a neutralising response in guinea pigs. However, the total number of HCMV-specified glycoproteins remains uncertain and the vaccine potential of individual glycoprotein is unknown. Purification of individual glycoproteins from HCMV-infected cells is a daunting prospect because the virus grows slowly and fails to shut down host cell protein synthesis during infection.
The present invention is based on the identification and expression of HCMV DNA encoding two glycoproteins, referred to herein as gB and gH. The gB protein is encoded by DNA in the HindIII F fragment of the HCMV genome lying between 1378 and 4095 bases from the F/D boundary. The gH protein is encoded by DNA in the HindIII L fragment lying between 228 and 2456 bases from the L/D boundary.
According to one aspect of the present invention there is provided a process which comprises expressing from a recombinant DNA vector in a suitable host organism a polypeptide incorporating one or more antigenic determinants capable of rising HCMV-neutralising antibodies in humans, said determinant or determinants corresponding to a portion of the protein encoded by DNA in the HindIII F fragment of the HCMV genome lying between 1378 and 4095 bases from the F/D boundary and/or a portion of the protein encoded by DNA in the HindIII L fragment of the HCMV genome lying between 228 and 2456 bases from the L/D boundary.
A second aspect of the present invention provides a recombinant virus vector containing DNA encoding such a polypeptide, said vector being capable of infecting a human subject and expressing the polypeptide in immunogenic form.
A third aspect of the present invention provides a process which comprises synthesising such a polypeptide.
A fourth aspect of the present invention provides a method of preparing HCMV monospecific antiserum comprising immunising a host animal with such polypeptide or with a recombinant virus vector as described above, and extracting from the host animal antiserum specific to said polypeptide. HCMV-specific monoclonal antibody may be prepared from cells from such immunised animals.
A fifth aspect of the present invention provides a method of purifying HCMV-specific antibodies, which comprises contacting the antibodies with HCMV polypeptide hereof, and separating bound antibody from the polypeptide.
A sixth aspect of the present invention provides a method of detecting HCMV-specific antibody in a clinical sample, which comprises contacting the sample with HCMV polypeptide hereof, and detecting antibody that binds to the polypeptide.
A seventh aspect of the present invention provides a kit for carrying out such a detection method, the kit comprising said polypeptide in a form suitable for contacting with the clinical sample, and means for detecting HCMV-specific antibody that binds to said polypeptide.
By identifying surface glycoprotein(s) of HCMV that lead to an immune response and incorporating the corresponding sequence of genetic material in a mammalian vector, an immunologically active protein may be produced which can form the basis of a vaccine against HCMV.
For the recombinant virus vaccine the identified HCMV genome fragment may be isolated and introduced into a suitable mammalian virus vector by conventional genetic engineering techniques, and transfecting the plasmid into a mammalian host.
Suitable vectors include mammalian cells and viruses such as poxviruses, with vaccinia virus being particularly preferred, and bovine papilloma virus.
Expression of the foreign DNA can be obtained by infecting cells or animals with recombinant virus vector. For example, a recombinant virus, e.g. vaccinia virus, may be used as a live vaccine. Further, cells infected with the recombinant vector may be used to prepare the product of the foreign DNA for use as a vaccine.
In one preferred technique, a glycoprotein-encoding fragment of the HCMV genome is introduced into plasmid pGS62 and then transferred into vaccinia virus by transfecting the plasmid into mammalian cells infected with vaccinia virus.
It will be apparent that the HCMV DNA may be modified in various ways without significantly affecting the functioning of the protein produced thereby. For example, a transmembrane form of protein may be converted to a secreted form by removing the DNA coding for the C-terminal containing the membrane anchor sequence. Such modifications are to be considered within the scope of the present invention.